Transcriptome and proteome analysis of dogs with precursor targeted immune-mediated anemia treated with splenectomy

Precursor-targeted immune-mediated anemia (PIMA) in dogs is characterized by persistent non-regenerative anemia and ineffective erythropoiesis, and it is suspected to be an immune-mediated disease. Most affected dogs respond to immunosuppressive therapies; however, some are resistant. In this study, we carried out splenectomy as an alternative therapy for refractory PIMA in dogs, and analyzed gene expression levels in the spleen of dogs with or without PIMA and in serum before and after splenectomy. A total of 1,385 genes were found to express differentially in the spleens from dogs with PIMA compared with healthy dogs by transcriptome analysis, of which 707 genes were up-regulated, including S100A12, S100A8, and S100A9 that are linked directly to the innate immune system and have been characterized as endogenous damage-associated molecular patterns. Furthermore, immunohistochemistry confirmed that S100A8/A9 protein expression levels were significantly higher in dogs with PIMA compared with those in healthy dogs. A total of 22 proteins were found to express differentially between the serum samples collected before and after splenectomy by proteome analysis, of which 12 proteins were up-regulated in the samples before. The lectin pathway of complement activation was identified by pathway analysis in pre-splenectomy samples. We speculated that S100A8/9 expression may be increased in the spleen of dogs with PIMA, resulting in activation of the lectin pathway before splenectomy. These findings further our understanding of the pathology and mechanisms of splenectomy for PIMA.

Introduction Non-regenerative immune-mediated anemia (NRIMA) [1,2] and precursor targeted immune-mediated anemia (PIMA) [3][4][5][6][7] have been described in dogs with non-regenerative anemia and evidence of ineffective erythropoiesis. There are currently no clear criteria for differentiating between these two disease conditions, and both diseases show similar responses against immunosuppressive therapy. Recent studies of PIMA examined dogs with few signs of peripheral immune-mediated destruction (spherocyte 3%, positive saline agglutination test 1.5% [4]). NRIMA is considered to be a broader disease, and studies looking at NRIMA often include dogs with symptoms of immune-mediated hemolytic anemia (IMHA) (spherocyte 19%, positive saline agglutination test 54% [2]). Terminology and diagnostic criteria often vary between reports: in this study we use the term PIMA according to the inclusion criteria previously reported [3][4][5][6][7][8], since the dogs in this study did not have any concurrent IMHA symptoms. With reference to treatments, 50%-88% of dogs with NRIMA/PIMA respond to immunosuppressive therapies, some dogs fail to respond to the treatments [1][2][3][4]. Intriguingly, splenectomy has been recommended as an alternative treatment for refractory immune-mediated hematopoietic diseases in humans and dogs [8][9][10][11][12]. We previously found that 75% of dogs refractory to immunosuppressive therapy responded to splenectomy [13]. Spleen-specific plasma cells producing anti-platelet antibodies were reported to be responsible for the improvement in immune-mediated thrombocytopenia after splenectomy in humans; however, there have been no similar veterinary studies to determine the mechanism of splenectomy in animals [14]. We therefore carried out transcriptome analysis of the spleen and proteomic analysis of pre-and post-splenectomy serum samples from dogs with PIMA, to clarify the mechanisms responsible for the beneficial effects of splenectomy in dogs with PIMA.

Ethics statement
Spleen and serum samples were obtained from client-owned dogs for veterinary diagnostic and treatment purposes with informed consent and the permission of the Ethics Screening Committee, Veterinary Teaching Hospital, Hokkaido University (permission number 2022-003). As controls, we used spleen samples that had been collected and preserved from healthy dogs that were euthanized in another study approved by the Hokkaido University Faculty of Veterinary Medicine Institutional Animal Care and Use Committee (#20-0081).

Study population
This study included 21 client-owned dogs diagnosed with PIMA that were presented to the Department of Internal Medicine at the Veterinary Teaching Hospital of Hokkaido University. The inclusion criteria were as follows: (1) minimum 5-day history of severe non-regenerative anemia (hematocrit <30%) with an absolute reticulocyte count <60×10 3 /μL [1,15]; (2) bone marrow cytology and/or histopathology results available with a diagnosis of ineffective erythropoiesis, defined as the presence of an erythropoietic response pattern (i.e., erythroid hypercellularity or increase in early-stage erythroid precursors with maturation arrest) not attributable to a pre-regenerative response or other disease process (e.g., myelodysplastic disease or drug reaction); (3) complete blood count, serum biochemistry profile, and thoracic and abdominal imaging results revealing no underlying cause for severe non-regenerative anemia; and (4) splenectomy performed because the animals had poorly responded to immunosuppressive therapy for >2 months, or because the animals were unable to receive immunosuppressive therapy due to suspected infection.
We also included three healthy dogs and three dogs without PIMA that underwent splenectomy because of a primary complaint of a spleen mass. Details of all the dogs are summarized in S1 Table.

Spleen samples
The histopathology of the spleen samples was evaluated by a member of the American College of Veterinary Pathologists (YK). Spleens from the healthy control dogs showed no significant changes, but 3 non-PIMA and 21 PIMA spleen samples showed extramedullary hematopoiesis. Splenic masses were diagnosed as hematoma and adipose nodules. Spleen samples were collected and stored in 10% neutral buffered formalin or RNAlater (Qiagen, Hilden, Germany) buffer at −80˚C for further histopathological and molecular analyses.

Serum samples
Serum samples were collected only from dogs with PIMA. Blood samples were collected before and after splenectomy by jugular venipuncture into tubes containing a coagulation activator and a gel separator. The tubes were then kept at room temperature until visible clotting had occurred. The samples were then centrifuged at 1,000 × g for 10 min and the serum was separated and stored at −80˚C until analysis. Pre-splenectomy serum samples were collected before blood transfusion. Post-splenectomy serum samples were collected approximately 2 months after surgery to eliminate the invasive effects of surgery, which may have affected the results.

Transcriptome analysis
Total RNA was extracted from PIMA and normal spleen tissues using an RNeasy Mini Kit (Qiagen). RNA integrity was examined using an Agilent 2100 Bioanalyzer (Agilent Technologies Japan, Tokyo, Japan), and the RNA integrity number of the total RNA isolated from each spleen was >8.5. Sequencing libraries were prepared using a QuantSeq 3 0 mRNA-Seq Library Prep Kit for Illumina (FWD) (Lexogen GmbH, Wien, Austria). RNA sequencing (75-bp single-end) was conducted with NextSeq 500 (Illumina, San Diego, CA, USA) using a NextSeq 500/550 High Output Kit v2.5 Kit (Illumina), and a minimum of 2.1 million reads were generated for each sample. Quantified read counts and differentially expressed genes (DEGs) were determined using RaNa-Seq (a bioinformatics tool for the analysis of RNA-seq data; https:// ranaseq.eu/index.php). FASTQ files were pre-processed with the Fastp tool [16], and expression was quantified using Salmon [17] with reference genome using Can_Fam_3.1. RNA sequences have been deposited in the repository of the DNA Data Bank of Japan with the accession number DRA015678. Differential expression was analyzed using DESeq2, and DEGs were determined based on an adjusted p-value of < 0.05 and log2 fold change (FC) > |2.0|. The comparison of distinct gene expression patterns was visualized in a principal component analysis (PCA) and volcano plots using R [18] and RJSplot [19].
Quantitative analysis for IHC samples S100A8/A9 stained slides were analyzed with QuPath ver 3.2.0 [20]. Three 1 mm 2 areas containing one lymph nodule were selected in each slide for S100A8/A9 signal counting. Hematoma and other lesions disrupting normal splenic structures were excluded from the analysis. To avoid the edge effects, areas within 500 μm from the tissue edge were not used. Cell segmentation was performed using StarDist extension for QuPath based on optical sum density values [21]. S100A8/A9 positive signals were distinguished from hemosiderin and formalin pigments at annotation steps. Annotation was performed every slide. The average number of positive cells per 1 mm 2 area were used for further analyses.

Serum proteome analysis
Albumin was removed from eight serum samples using a ProMax albumin removal kit (Bangs Laboratories Inc., Fishers, IN, USA). Ten microliters of serum was added to 35 μL of ProMax Binding/Wash Buffer and 50 μL of ProMax particles, followed by gentle mixing for 20 min at room temperature. The particles were then collected by magnetic separation and the supernatant containing albumin was removed. The particles were washed three times with 500 μL Pro-Max Binding/Wash Buffer and then mixed for 10 min at room temperature in 80 μL of 100 mM Tris-HCl pH 8.5 and 0.5% sodium dodecanoate. The supernatant was transferred to a fresh 1.5 mL tube after magnetic separation of the particles, 20 μL of the sample was treated with 10 mM dithiothreitol at 50˚C for 30 min, followed by alkylation with 30 mM iodoacetamide in the dark at room temperature for 30 min. The reaction was stopped with 60 mM cysteine for 10 min. The mixture was then diluted with 150 μL of 50 mM Tris-HCl pH 8.0 and digested by adding 400 ng of Trypsin/Lys-C mix (Promega, Madison, WI, USA) overnight at 37˚C. The digested sample was acidified with 30 μL of 5% trifluoroacetic acid followed by sonication (Bioruptor UCD-200, CosmoBio, Tokyo, Japan) for 5 min. The mixture was shaken for 5 min and centrifuged at 15,000 × g for 5 min and the supernatant was desalted using C18-StageTips [22], followed by drying using a centrifugal evaporator. The dried peptides were redissolved in 3% acetonitrile and 0.1% formic acid measured using a colorimetric peptide assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and transferred to a hydrophilic-coated low-adsorption vial (ProteoSave vial; AMR Inc., Tokyo, Japan).
For liquid chromatography (LC) separation, the mobile phases consisted of 0.1% (v/v) formic acid as solvent A and 0.1% (v/v) formic acid/80% (v/v) acetonitrile as solvent B. Each peptide sample (200 ng) was injected directly onto a 75 μm × 12 cm nanoLC nano-capillary column (Nikkyo Technos Co., Ltd., Tokyo, Japan) at 40˚C and then separated with a 40 min gradient at a flow rate of 150 nL/min using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific). Peptides eluted from the column were analyzed using a Q Exactive HF-X (Thermo Fisher Scientific) for overlapping window DIA-MS [23,24]. Mass spectrometry (MS) 1 spectra were collected in the range of 495-785 m/z at 30,000 resolution to set an automatic gain control target of 3e6 and maximum injection time of 55. MS2 spectra were collected in the range >200 m/z at 15,000 resolution to set an automatic gain control target of 3e6, maximum injection time of "auto", and a normalized collision energy of 28%. The isolation width for MS2 was set to 4 m/z and overlapping window patterns in 500-780 m/z were used window placements optimized by Skyline v4.1 [25].
MS files were searched against a dog spectral library using Scaffold DIA (Proteome Software, Inc., Portland, OR, USA). The dog spectral library was generated from dog protein sequence database (Proteome ID: UP000002254) by Prosit [26,27]. The Scaffold DIA search parameters were as follows: experimental data search enzyme, trypsin; maximum missed cleavage sites, 1; precursor mass tolerance, 8 ppm; fragment mass tolerance, 8 ppm; and static modification, cysteine carbamidomethylation. The protein identification threshold was set for both peptide and protein false discovery rates (FDRs) of <1%. Peptide quantification was calculated using the EncyclopeDIA algorithm [28] in Scaffold DIA. For each peptide, the four highest quality fragment ions were selected for quantitation. Protein quantification was estimated from the summed peptide quantification. We only used proteins that met the criteria of a peptide FDR <1%, protein FDR <1%, and number of peptide fragments �2. The proteomic datasets have been deposited into the repository of ProteomeXchange and Japan ProteOme STandard Repository with the accession numbers PXD039913 and JPST002024 respectively. The thresholds for altered proteins were p < 0.05 (t-test) and fold change >2 or <0.5 between the pre-and post-splenectomy groups.

Pathway analysis
DEG-and differentially expressed protein-related pathways were analyzed with PANTHER 17.0 (http://www.pantherdb.org/), using the annotation dataset "Reactome pathway" to identify the activated pathways. We only included significantly (p < 0.05) up-or down-regulated pathways in our results.

Transcriptome analysis and pathway analysis
We analyzed spleen samples from 15 dogs with PIMA and three healthy dogs to identify PIMA-related DEGs in the spleen. A total of 15,903 genes were detected by transcriptome analysis. The gene expression profiles were analyzed by PCA (Fig 1), which clearly separated normal spleen from PIMA samples.
We identified a total of 1,385 DEGs between spleen samples from normal dogs and dogs with PIMA (8.7% of all identified genes), with a p-value < 0.05. Of these, 707 genes were upregulated and 678 were down-regulated in dogs with PIMA (Fig 2). The top 10 over-and under-expressed genes are shown in Table 1, and all DEGs are shown in S2 Table. We then performed pathway analysis of the 300 most strongly up-and down-regulated DEGs. The top 10 pathways enriched in the up-regulated genes were included in "Cell Cycle" and "DNA Replication", possibly because extramedullary hematopoiesis was detected in all PIMA dogs but not in the normal dogs by histopathologic examination. The top 10 over-and under-expressed pathways are shown in Table 2 and S3 Table. IHC for S100A8/A9 Transcriptome analysis revealed that S100A12, S100A8, and S100A9 were the top overexpressed genes in PIMA spleen (p < 9.6E-11, 2.9E-10, and 6.8 E-10; Table 1). We specifically

PLOS ONE
Molecular effects of splenectomy in dogs with refractory precursor-targeted immune-mediated anemia focused on S100A8/A9, which are also known to play a role in erythrocyte hematopoiesis and complement activation [29,30]. We verified the protein expression levels and localization of S100A8/A9 in spleen samples from 21 dogs with PIMA, three healthy dogs, and three non-PIMA dogs, to investigate the relationship between S100A8/A9 and extramedullary hematopoiesis. Few S100A8/A9-positive cells were observed at the white pulp margin in healthy dogs and non-PIMA dogs, while numerous positive cells were observed in the medullary red pulp as well as the white pulp in dogs with PIMA (Fig 3). The number of S100A8/A9-positive cells was significantly higher in PIMA spleen compared with healthy and non-PIMA spleen samples (p = 0.0412 and p = 0.0412, respectively) (Fig 4).

Differentially expressed proteins and pathways in pre-and postsplenectomy serum samples in dogs with PIMA
We performed proteomic profiling of serum samples using LC tandem mass spectrometry to reveal the physiological changes between pre-and post-splenectomy. We obtained pre-and post-splenectomy samples from four dogs with PIMA (8 samples) and identified 432 proteins in the eight serum samples, of which 342 proteins met the analysis criteria (S4 Table). A total of 22 proteins were differentially expressed between the pre-and post-splenectomy serum  (Table 3). Twelve proteins were down-regulated and 11 were up-regulated in the post-splenectomy compared with the pre-splenectomy samples. Finally, we focused on the enriched pathways related to 22 significantly altered serum proteins. The Reactome pathway "ficolins bind to repetitive carbohydrate structures on the target cell surface" was significantly up-regulated in pre-splenectomy samples (p = 4.91E-06) (S5 Table). This pathway included ficolin 1 (FCN1) and mannan-binding lectin serine peptidase 2 (MASP2), which were up-regulated 4-and 2.8-fold, respectively, in pre-compared with postsplenectomy samples.

Discussion
In this study, we investigated the mechanisms responsible for the effects of splenectomy in dogs with PIMA by transcriptome analysis of spleen samples and proteomics analysis of preand post-splenectomy serum samples. Based on the results of transcriptome analysis, we validated the protein expression of S100A8/A9 in spleen samples using IHC.
In the transcriptome analysis, PCA of 15,903 genes showed a clear separation between spleen samples from dogs with PIMA and healthy dogs, suggesting that PIMA may significantly affect gene expression in the spleen. Among these, 1,385 genes were detected as DEGs, of which the top three most highly overexpressed genes in PIMA spleens were S100A12,   S100A8, and S100A9. S100A8/A9 protein was also highly expressed, especially in PIMA samples, as shown by IHC. The S100 protein family comprises the largest subgroup within the Ca 2 + -binding EF-hand protein superfamily [31]. S100A8/A9 and S100A12 are released from activated monocytes and granulocytes and act as proinflammatory endogenous Toll-like receptor 4-ligands [32][33][34]. Serum levels of S100A12 and S100A8/A9 were shown to be increased in various inflammatory and autoimmune diseases, and complement activation was reported to occur at sites expressing S100A8/A9 [30,31,[35][36][37]. Previous studies have suggested that PIMA may be an autoimmune disease [6], which may partially explain the elevated S1008/A9 levels.

PLOS ONE
One of the significant clinicopathological findings is the presence of ineffective erythropoiesis in dogs with PIMA. S100A9 was also shown to suppress erythroid differentiation in both experimentally induced deletion 5q subtype of myelodysplastic syndrome through inactivation of Rps14 and wild-type mouse model, specifically the hematopoietic stem cells and progenitor cells [29]. Moreover, serum levels of S100A8 were significantly elevated in patients with myelodysplastic syndrome compared with healthy controls [38]. Thus, the inhibitory effect of S100A9 on erythroblast differentiation may be responsible for the ineffective erythropoiesis in PIMA dogs.
We also carried out proteomic analysis of pre-and post-splenectomy serum samples and found that 22 proteins were differentially expressed. Bactericidal/permeability-increasing foldcontaining family B member 4 (BPIFB4), haptoglobin, and FCN1 were specifically down-regulated in post-splenectomy compared with pre-splenectomy serum samples. BPIFB4 serum levels were 26-fold higher before compared with after splenectomy, while its RNA expression levels were significantly higher in PIMA spleens than those in healthy spleens (p = 0.003). Given these results, BPIFB4 may be produced in the spleen, and may be expressed at higher levels in dogs with PIMA. The concentration of BPIFB4 in serum appears to then decrease after a splenectomy. However, these data are inconsistent with the fact that BPIFB4 is typically more abundant in the serum of healthy, long-lived individuals compared with frail individuals [39]. This has been postulated to be due to advantageous anti-inflammatory effects through macrophage polarization [40] and correction of hypertension in humans [41]. Unfortunately, there is insufficient information to understand the direct involvement of BPIFB4 in nonregenerative anemia, and its association with the pathogenesis of PIMA remains unknown.
A study that investigated the clinical progression of horses after splenectomy found that two months after the procedure, the concentration of haptoglobin was significantly reduced compared with those that had not been splenectomized [42]. Our findings are consistent with these data, and the collection timelines were similar because we collected post-splenectomy serum samples from the dogs more than 2 months after the procedure. However, haptoglobin levels can be influenced by the administration of corticosteroids to dogs, and increased haptoglobin levels are often observed after corticosteroid therapy [43,44] and in spontaneous hyperadrenocorticism [45]. Together, these data suggest that changes in serum haptoglobin could be affected by the splenectomy; however, we cannot exclude the possibility of elevated haptoglobin due to corticosteroid therapy.
The reactome pathways "Lectin pathway of complement activation" were over-represented in our list of pathways upregulated before splenectomy. The complement system can be activated through three major pathways, including the classical, lectin, and alternative lectin pathways. Complement activation, regardless of the pathway, ultimately triggers three effector pathways (MAC assembly, anaphylatoxins, and opsonization) that enable the complement to fulfill its physiological role in host defense. The lectin pathway is initiated by binding of pattern-recognition molecules, including FCN, and this pathway is accelerated by MASP, which was upregulated in pre-splenectomy serum [46]. FCN1 is a multimeric protein consisting of an N-terminal collagen-like domain and a C-terminal fibrinogen-like domain, which is present in secretory granules and is primarily expressed in granulocytes and monocytes. Serum levels of FCN1 are elevated in vasculitis syndrome and arthritis, and FCN1 has thus gained attention as a potential therapeutic target in autoimmune diseases [47]. FCN1 RNA expression was also up-regulated in the peripheral blood in dogs with IMHA [48]. In the present study, FCN1 and MASP2 were up-regulated 4-and 2.8-fold, respectively, in pre-compared with post-splenectomy serum, suggesting that uncontrolled non-regenerative anemia with complement activation may be relevant to the immune-mediated destruction of erythroid progenitor cells, which has been considered as a pathophysiology of PIMA [6]. However, FCN1 expression data could not be obtained in our transcriptome analysis, and MASP2 expression levels were similar between samples from dogs with PIMA and healthy dogs, despite their significant upregulation in serum before the splenectomy. MASP2 is reported to be produced mainly in the liver [49], and FCN1 is located primarily in peripheral blood [50,51], suggesting that these genes are not highly expressed in the spleen, which may explain this discrepancy between the transcriptomic and proteomic data. Nevertheless, since FCN1 and MASP2 are involved in the lectin pathway that induces the activation of complement pathway [52], we speculate that the changes in levels of these proteins in the serum may be affected by the release of a specific factor, such as S100A8/A9 (Fig 5).
Considering the overall mechanism prior to splenectomy in dogs with PIMA, we predicted that S100A8/9 would be increased in the spleen in PIMA dogs, resulting in activation of the lectin pathway ( Fig 5). However, the reason behind the up-regulation of S100A8/A9 in the spleen remains unknown. Expression levels of S100A8 and S100A9 in various organs have been reported to increase with ageing [53]. PIMA is most commonly reported in middle-aged and older dogs, but a previous study reported a median onset age of 6.5 years [4]. In contrast, the median age in the current group was 12 years, which was similar to a retrospective study of Miniature Dachshunds with suspected PIMA in Japan, which reported a median age of 10.9 years [54]. Despite the differences in median age compared with Assenmacher et al.'s study [4], we speculated that there might be an association between the characteristic clinical course of PIMA and the high expression of S100A8/A9 because the dogs in these three studies were older than 6.5 years.
There are several limitations to this study. Insufficient samples from both non-PIMA and healthy dog groups due to difficulty in obtaining samples. Majority of samples were MDs though this is the consistency of the previous report [4]. Pathophysiology may differ depending on treatment responsiveness to splenectomy. Further large-scale studies in specific breeds and organs and in different treatment settings are needed to identify the precise pathology of PIMA in dogs.